The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are effective therapeutic agents for patients with non-small cell lung cancer (NSCLC) whose tumors harbor activating mutations in EGFR. Most, if not all, patients who initially develop a partial or complete response to gefitinib or erlotinib will eventually develop progression of their cancer while taking these therapies. The only known mechanism of resistance, a secondary mutation in EGFR itself (a substitution of methionine for threonine at position 790, EGFR T790M) has been detected in approximately 50% of patients developing resistance to gefitinib or erlotinib. This finding has spurred the clinical development of irreversible EGFR inhibitors that can inhibit an EGFR T790M to treat cancers that have become resistant to gefitinib/erlotinib. To identify of mechanisms of acquired resistance to gefitinib or erlotinib, we have generated gefitinibresistant clones of EGFR mutant NSCLC cell lines by exposing them to increasing concentrations of gefitinib. In previous work, we identified an EGFR T790M in vitro model of resistance, thereby demonstrating that in vitro models can be used to discover resistance mechanisms observed in patients. These paired [parental and resistant clone) cell lines provide valuable preclinical models in which to systematically determine mechanisms of gefitinib resistance and their in vitro sensitivity to novel therapeutic agents. Once dentified, tumor specimens from EGFR mutant patients that have developed acquired resistance to gefitinib/erlotinib will be assessed to determine if these resistance mechanisms can also be detected in aatients. Furthermore, based on the findings above, novel therapeutic combinations will be evaluated in the gefitinib-resistant cell line models with differing mechanisms of resistance. These studies will serves as the jasis for rationally designed clinical trials for patients with gefitinib/erlotinib resistance. These studies will be accomplished through the following specific aims: Aim 1: To discover mechanisms of acquired resistance to EGFR-targeted agents. Aim 2: To determine whether targeting resistance mechanisms in vitro using pharmacologic inhibitors or by RNA interference (RNAi) will lead to growth inhibition of resistant NSCLC cell ines Aim 3:To design and conduct clinical studies in NSCLC patients with different mechanisms of acquired resistance to gefitinib/erlotinib. Since EGFR TKIs are highly effective initial treatments for patients with EGFR mutant cancers, it will be critical to identify the how these cancers eventually become resistant. The studies n this grant proposal will help develop the future treatments for patients with different mechanisms of acquired resistance to EGFR TKIs.